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Mouse NS-1 myeloma cells were fused with spleen cells from mice that had been immunized with cells from a human melanoma, M1804. Hybrid cells were grown in selective medium and tested for production of antibody to surface antigens of M1804 cells. Three hybrids that produced antibodies that bound to the melanoma cells but not to autologous skin fibroblasts were cloned. Antibodies produced by two of the clones were cytotoxic to M1804 cells in the presence of rabbit complement. Extensive specificity tests showed that the antibodies produced by the clones bound strongly only to M1804 cells; significant, although weaker, binding occurred with 2 of 11 allogeneic melanomas. Apart from weak binding of the antibody produced by one of the clones to a breast carcinoma, binding assays of five carcinomas, one sarcoma, and fibroblasts from 17 individuals were negative, as were cytotoxic tests of 10 lymphoblastoid cell lines and peripheral blood lymphocytes from 68 normal donors and 12 chronic lymphocytic leukemia patients. This suggests that we have identified one or more determinants of a melanoma-associated antigen(s), whose expression is limited to a small proportion of melanomas. Human melanomas express cell surface antigens to which melanoma patients can mount an immune response (1). Sera from such patients sometimes contain antibodies to cell surface antigens of their melanomas. In some cases the antigens have been detected only on the patient's own melanoma (2, 3), and in other cases, also on allogeneic melanomas (3-5). There is also evidence for cell-mediated immunity, both to antigens whose expression is limited to a small number of melanomas (6) and to antigens that are shared by most, or possibly all, melanomas (7-9).The introduction by Kohler and Milstein (10) (see also ref. 11) of a method for producing large amounts of monoclonal antibodies of defined specificity promises to revolutionize serological analysis of tumor antigens. Koprowski et al. (12) have used this approach and obtained a monoclonal mouse antibody that identifies what may be a melanoma-specific antigen.This paper reports the results of a fusion of mouse NS-1 myeloma cells with spleen cells from mice that had been immunized with a short-term explant of a human melanoma (M1804). Three of the cell hybrids produced antibodies that bound to the immunizing melanoma, but not to autologous skin fibroblasts. These hybrids have been cloned and tested extensively by means of a '2-I-labeled protein A (12'I-protein A) binding assay and complement-dependent cytotoxicity assays. Results have shown that the antibodies produced by the clones define antigenic determinants expressed strongly on M1804 melanoma cells and weakly on some allogeneic melanomas, but not appreciably on other human cell types. MATERIALS AND METHODSCells. M1804 is a short-term explant of a melanoma metastasis that was removed from a lymph node of a 51-yr old white male 6 months after excision of the primary tumor (superficial spreading L III). Skin fibroblasts from the same patient...